Experiments are proposed to investigate the mechanism of ischemic preconditioning (PC), a powerful method of protecting myocardium from infarction during ischemia. During the past funding period we found that protein kinase C (PKC) is intimately involved, in PC. We proposed that exposure of the heart to a brief period of ischemia liberates adenosine which occupies adenosine receptors which are coupled to PKC and that normally this pathway is down-regulated. The brief ischemic exposure initiates an up-regulation of the pathway (probably by translocating PKC into the membranes). When a second period of ischemia occurs reoccupation of adenosine receptors results in an immediate phosphorylation of PKC substrate since PKC is already up-regulated. The data indicate that protection will occur only if there is no delay in PKC activation at the onset of ischemia. The present application will critically test our translocation hypothesis. The hypothesis predicts that any receptor coupled to PKC should be capable of putting the heart into the PC state and specific aim 1 will test that prediction with phenylephrine, angiotensin and endothelin. In aim 2 we will actually monitor PKC activity by looking at phosphorylation of MARCKS protein. In aim 3 we will test whether we can pharmacologically PC isolated rabbit myocytes. Aim 4 will examine the phospholipase coupling between adenosine receptors and PKC to see if phospholipase D might be involved. We find that rabbits quickly become tolerant to adenosine agonists or even to repeated bouts of ischemic PC such that they are no longer protective. We will determine the level at which tolerance develops in aim 5. Aim 6 will attempt to validate the translocation theory by localizing PKC isoforms in the cell with immunocytochemistry. Finally aim 6 will test whether PKC is involved in PC in the rat heart.